Development of an AI-Assisted Embryo Selection System Using Iberian Ribbed Newts for Embryo-Fetal Development Toxicity Testing.

Background: The 3Rs (Reduction, Refinement, Replacement) principle is driving the need for alternative methods in animal testing. Despite advancements in in vitro testing, complex systemic toxicity tests still necessitate in vivo approaches. The aim of this study was to develop a developmental toxicity test protocol using the Iberian ribbed newt (Pleurodeles waltl) as a model organism, integrating AI image analysis for embryo selection to improve test accuracy and reproducibility. Methods: We established a developmental toxicity test protocol based on the zebrafish test. Gonadotropin was administered to induce ovulation, and in vitro fertilization was performed. Embryos were imaged at 5-6 and 6-7 h post-fertilization. AI image analysis was utilized to assess embryo viability. The test chemical was administered 24-48 h post-fertilization, and morphological changes were observed daily until day 8. Additionally, a time-lapse photography system was constructed to monitor embryonic development. Results: Out of 24 cultured embryos, 75% developed normally to the late tail bud stage or initial hatching stage, whereas 25% experienced developmental arrest or death. AI image analysis achieved high accuracy in classifying embryos, with overall accuracies of 92.0% and 92.9% for two learning models. The AI system demonstrated higher precision in the selection of viable embryos compared to visual inspection. Conclusion: The Iberian ribbed newt presents a viable alternative model for developmental toxicity testing, adhering to the 3Rs principles. The integration of AI image analysis substantially enhances the accuracy and reproducibility of embryo selection, providing a reliable method for evaluating developmental toxicity in pharmaceuticals.

Trophic effects of Bti-based mosquito control on two top predators in floodplain pond mesocosms.

Chironomid (Diptera: Chironomidae) larvae play a key role in aquatic food webs as prey for predators like amphibian and dragonfly larvae. This trophic link may be disrupted by anthropogenic stressors such as Bacillus thuringiensis var. israelensis (Bti), a biocide widely used in mosquito control. In a companion study, we recorded a 41% reduction of non-target larval chironomids abundance in outdoor floodplain pond mesocosms (FPMs) treated with Bti. Therefore, we examined the diet of two top predators in the FPMs, larvae of the palmate newt (Salamandridae: Lissotriton helveticus) and dragonfly (Aeshnidae: predominantly Anax imperator), using bulk stable isotope analyses of carbon and nitrogen. Additionally, we determined neutral lipid fatty acids in newt larvae to assess diet-related effects on their physiological condition. We did not find any effects of Bti on the diet proportions of newt larvae and no significant effects on the fatty acid content. We observed a trend in Aeshnidae larvae from Bti-FPMs consuming a higher proportion of large prey (Aeshnidae, newt, damselfly larvae; ~42%), and similar parts of smaller prey (chironomid, mayfly, Libellulidae, and zooplankton), compared to controls. Our findings may suggest bottom-up effects of Bti on aquatic predators but should be further evaluated, for instance, by using compound-specific stable isotope analyses of fatty acids or metabarcoding approaches.

Integrative systematic revision of the Montseny brook newt (Calotriton arnoldi), with the description of a new subspecies.

The Montseny brook newt (Calotriton arnoldi), a glacial relict endemic to a small, isolated massif in northeast Spain, is considered the only Critically Endangered urodele in Europe. Its restricted range is divided by a deep valley that acts as an impassable barrier to dispersal, separating two isolated metapopulations (Western and Eastern) that correspond to independent lineages with different evolutionary trajectories, based on genetic and genomic data. Here, we address the ecological differentiation between lineages and discuss its potential effect on the phenotypic distinctness of each lineage. Based on multiple lines of evidence, we formally describe the Western Montseny brook newt as a new subspecies: Calotriton arnoldi laietanus ssp. nov. Finally, our study underscores the importance of considering taxonomic progress in the conservation policies of endangered species, ensuring appropriate management and protection of the newly described taxa.

Transcriptome variation in banded newt (Ommatotriton vittatus) during its life cycle and habitat transition.

Israel represents the southern limit of the distribution of the banded newt (Ommatotriton vittatus). The life cycle of O. vittatus includes several distinct phases: eggs, aquatic larvae, a terrestrial phase and an aquatic reproductive phase. We investigated differences in gene expression during the life cycle and transition of banded newts between terrestrial and aquatic habitats using mRNA-seq. We identified ∼10 k genes that were differentially expressed (DE) in one of the pairwise comparisons between 3 groups: 1 - terrestrial newts (males and females), 2 - aquatic newts (males and females), 3 - aquatic larvae before metamorphosis. The groups were clearly defined by Principal Components Analysis (PCA). The greatest difference was between aquatic newts (males and females) and aquatic larvae: ∼7.4 k DE genes. Of special interest were the ∼2.4 k genes DE between the aquatic and terrestrial phenotypes. These included prominent candidates with known roles in kidney function (uromodulin homologs were strongly associated with aquatic lifestyle), tissue structure (keratins), and the thyroid hormone signaling modulator DUOXA1. Additional developmental and metabolic pathways overrepresented among the identified DE genes included "epidermis development", "nervous system development", "nucleotide-sugar biosynthesis". Overall, both metamorphosis and environmental transition of banded newts involve extensive transcriptomic remodeling involving developmental, metabolic, and cellular pathways. Understanding the roles of these pathways and individual genes is instrumental for studies of transition between habitats, especially those affected by climate change. Furthermore, the phenotypic flexibility of the newt and the underlying regulation of gene expression can shed light on the evolution of terrestrial vertebrates.

FGF-stimulated tendon cells embrace a chondrogenic fate with BMP7 in newt tissue culture.

Newts can regenerate functional elbow joints after amputation at the joint level. Previous studies have suggested the potential contribution of cells from residual tendon tissues to joint cartilage regeneration. A serum-free tissue culture system for tendons was established to explore cell dynamics during joint regeneration. Culturing isolated tendons in this system, stimulated by regeneration-related factors, such as fibroblast growth factor (FGF) and platelet-derived growth factor, led to robust cell migration and proliferation. Moreover, cells proliferating in an FGF-rich environment differentiated into Sox9-positive chondrocytes upon BMP7 introduction. These findings suggest that FGF-stimulated cells from tendons may aid in joint cartilage regeneration during functional elbow joint regeneration in newts.

Biomechanical analysis of tendon regeneration capacity of Iberian ribbed newts following transection injury: Comparison to a mouse model.

Adult mammals are known for their poor ability to regenerate tissues, including tendons. On the other hand, urodeles have become an important model in regenerative studies for their remarkable ability to regenerate various body parts and organs throughout life, such as limbs, retinas, or even the brain. However, little is known about their capacity to regenerate injured tendons. If newts can also repair tendons without scar formation, they may be a suitable animal model for tendon regeneration studies in other adult vertebrates. Therefore, the present study used Iberian ribbed newts to characterize mechanical and structural regeneration of tendons following transection, using tensile tests and multiphoton microscopy. A digital flexor tendon in a hindlimb was transected either partially or completely, and regenerated tendon was examined 6 and 12 weeks after the operation. Tensile strength of regenerated tendons was significantly less than normal at 6 weeks, but was remarkably recovered at 12 weeks, reaching levels comparable to those of uninjured tendons. On the other hand, mouse tendons demonstrated poor recovery of strength even after 12 weeks. Multiphoton microscopy revealed that tendon-like collagenous tissue bridges residual tendon stubs in newts, but disorganized scar-like tissue filled the injured location in mice. These findings highlight the remarkable capacity of newts to recover from tendon injury and confirm the utility of newts as a model to study tendon regeneration.

Molecular Signatures Integral to Natural Reprogramming in the Pigment Epithelium Cells after Retinal Detachment in Pleurodeles waltl.

The reprogramming of retinal pigment epithelium (RPE) cells into retinal cells (transdifferentiation) lies in the bases of retinal regeneration in several Urodela. The identification of the key genes involved in this process helps with looking for approaches to the prevention and treatment of RPE-related degenerative diseases of the human retina. The purpose of our study was to examine the transcriptome changes at initial stages of RPE cell reprogramming in adult newt Pleurodeles waltl. RPE was isolated from the eye samples of day 0, 4, and 7 after experimental surgical detachment of the neural retina and was used for a de novo transcriptome assembly through the RNA-Seq method. A total of 1019 transcripts corresponding to the differently expressed genes have been revealed in silico: the 83 increased the expression at an early stage, and 168 increased the expression at a late stage of RPE reprogramming. We have identified up-regulation of classical early response genes, chaperones and co-chaperones, genes involved in the regulation of protein biosynthesis, suppressors of oncogenes, and EMT-related genes. We revealed the growth in the proportion of down-regulated ribosomal and translation-associated genes. Our findings contribute to revealing the molecular mechanism of RPE reprogramming in Urodela.

Step-by-step protocol for alternative injury models in newt cardiac regeneration.

Although the heart is one of the most important organs for animal survival, its regenerative capacity varies among animal species. Notably, adult mammals cannot regenerate their hearts after damage such as acute myocardial infarction. In contrast, some vertebrate animals can regenerate the heart throughout their lives. Cross-species comparative studies are important to understand the full picture of cardiac regeneration in vertebrates. Among the animal species able to regenerate the heart, some urodele amphibians, such as newts, possess a remarkable capacity for this process. Standardized methods of inducing cardiac regeneration in the newt are needed as a platform for studies comparing newts and other animal models. The procedures presented here describe amputation and cryo-injury techniques for the induction of cardiac regeneration in Pleurodeles waltl, an emerging model newt species. Both procedures consist of simplified steps that require no special equipment. We additionally show some examples of the regenerative process obtained using these procedures. This protocol has been developed for P. waltl. However, these methods are also expected to be applicable to other newt and salamander species, facilitating comparative research with other model animals.

Histological changes of the skin during postembryonic development of the crested newt Triturus ivanbureschi (Urodela, Salamandridae).

BACKGROUND: Amphibian skin has been studied for many decades, especially the metamorphic changes in the skin of frogs. Less attention has been paid to salamander skin. Here, we describe changes in the skin structure during postembryonic development in a salamandrid species, the Balkan crested newt Triturus ivanbureschi. METHOD: Using traditional histological techniques we examined the skin in the trunk region of three premetamorphic larval stages (hatchling, mid larval and late larval) and two postmetamorphic stages (juvenile, just after metamorphosis, and adult). RESULTS: In larval stages, skin consists only of the epidermis, which gradually develops from the single epithelial cell layer in hatchlings, to a stratified epidermis with gland nests and characteristic Leydig cells at the late larval stage. During metamorphosis, Leydig cells disappear, and the dermal layer develops. In postmetamorphic stages, skin is differentiated on stratified epidermis and the dermis with well-developed glands. Three types of glands were observed in the skin of the postmetamorphic stages: mucous, granular and mixed. Gland composition appears to be stage- and sex-specific, with juveniles and adult female being more similar to each other. In juveniles and adult female, there are a similar proportion of glands in both dorsal and ventral skin, whereas in adult male granular glands dominated the dorsal skin, while mixed glands dominated the ventral skin. CONCLUSION: Our results provide a baseline for future comparative research of skin anatomy in salamanders.

A small noncoding RNA links ribosome recovery and translation control to dedifferentiation during salamander limb regeneration.

Building a blastema from the stump is a key step of salamander limb regeneration. Stump-derived cells temporarily suspend their identity as they contribute to the blastema by a process generally referred to as dedifferentiation. Here, we provide evidence for a mechanism that involves an active inhibition of protein synthesis during blastema formation and growth. Relieving this inhibition results in a higher number of cycling cells and enhances the pace of limb regeneration. By small RNA profiling and fate mapping of skeletal muscle progeny as a cellular model for dedifferentiation, we find that the downregulation of miR-10b-5p is critical for rebooting the translation machinery. miR-10b-5p targets ribosomal mRNAs, and its artificial upregulation causes decreased blastema cell proliferation, reduction in transcripts that encode ribosomal subunits, diminished nascent protein synthesis, and retardation of limb regeneration. Taken together, our data identify a link between miRNA regulation, ribosome biogenesis, and protein synthesis during newt limb regeneration.

Corrigendum: Electrolyte imbalances and dehydration play a key role in Batrachochytrium salamandrivorans chytridiomycosis.

[This corrects the article DOI: 10.3389/fvets.2022.1055153.].

CRISPR-Cas9-Based Functional Analysis in Amphibians: Xenopus laevis, Xenopus tropicalis, and Pleurodeles waltl.

Amphibians have made many fundamental contributions to our knowledge, from basic biology to biomedical research on human diseases. Current genome editing tools based on the CRISPR-Cas system enable us to perform gene functional analysis in vivo, even in non-model organisms. We introduce here a highly efficient and easy protocol for gene knockout, which can be used in three different amphibians seamlessly: Xenopus laevis, Xenopus tropicalis, and Pleurodeles waltl. As it utilizes Cas9 ribonucleoprotein complex (RNP) for injection, this cloning-free method enables researchers to obtain founder embryos with a nearly complete knockout phenotype within a week. To evaluate somatic mutation rate and its correlation to the phenotype of a Cas9 RNP-injected embryo (crispant), we also present accurate and cost-effective genotyping methods using pooled amplicon-sequencing and a user-friendly web-based tool.

Salamanders as Key Models for Development and Regeneration Research.

For 70 years from the very beginning of developmental biology, the salamander embryo was the pre-eminent model for these studies. Here I review the major discoveries that were made using salamander embryos including regionalization of the mesoderm; patterning of the neural plate; limb development, with the pinnacle being Spemann's Nobel Prize for the discovery of the organizer; and the phenomenon of induction. Salamanders have also been the major organism for elucidating discoveries in organ regeneration, and these are described here too beginning with Spallanzani's experiments in 1768. These include the neurotrophic hypothesis of regeneration, studies of aneurogenic limbs, the concept of dedifferentiation and transdifferentiation, and advances in understanding pattern formation via molecules located on the cell surface. Also described is the prodigious power of brain and spinal cord regeneration and discoveries from lens regeneration, all of which reveal how important salamanders have been as research models.

Motilin is a regulator of gastric contraction in Japanese fire belly newts (Cynops pyrrhogaster), in vitro studies using isolated gastrointestinal strips of newts, rabbits, and chickens.

The effects of newt motilin on the contractility of the isolated gastrointestinal (GI) tract from Japanese fire belly newts (newt) were examined to clarify whether motilin regulates GI motility in urodele amphibians. In addition, contractile responsiveness to motilins from seven species of vertebrates (human, chicken, turtle, alligator, axolotol, newt and zebrafish) were compared in GI preparations from three different animals (rabbit duodenum, chicken ileum and newt stomach) to determine the species-specific action of motilin. Newt motilin (10-10 M - 10-6 M) caused a contraction of cognate gastric strips, while the upper, middle, and lower intestinal strips were insensitive. The rank order of motilins for contractile activity in newt gastric strips was newt > alligator > axolotol > chicken > turtle > human ≫ zebrafish. On the other hand, newt motilin caused a weak contraction in the rabbit duodenum (human > alligator = chicken > turtle > newt ≧ axolotol > zebrafish), and it was ineffective in the chicken ileum (chicken > turtle > alligator > human ≫ newt, axolotol and zebrafish). This study demonstrates that motilin induces contraction in the GI tract of a urodele amphibian, the newt, in a region (stomach)-specific manner and further indicates that a ligand-receptor interaction of the motilin system is a species-specific manner probably due to differences in the amino acid sequence of motilin.

Southbound - the southernmost record of Tylototriton (Amphibia, Caudata, Salamandridae) from the Central Highlands of Vietnam represents a new species.

A new species of the genus Tylototriton is described from Ngoc Linh Mountain, Kon Tum Province, in the Central Highlands of Vietnam based on integrative taxonomy, namely by combining molecular and morphological evidence. Tylototritonngoclinhensissp. nov. differs from all other congeners based on morphological data, allopatric distribution, and molecular divergence. In terms of genetic divergence, Tylototritonngoclinhensissp. nov. distinctly differs from the sister species T.panhai (6.77%) and from T.ngarsuensis (12.36%) based on the mitochondrial NADH dehydrogenase subunit 2 (ND2) gene. Tylototritonngoclinhensissp. nov. is a moderate sized and robust salamander species with large cephalic edges, parotoids, and vertebral ridge orange in coloration. The new taxon differs from its congeners by a combination of the following morphological characteristics: size medium (SVL 60.8-66.5 mm, TL 57.6-61.8 mm in males, and SVL 72.5-75.6 mm, TL 62.9-67.9 mm in females); head longer than wide; parotoids very prominent and enlarged, projecting backwards; tail length shorter than snout-vent length; vertebral ridge large, high and glandular in appearance; 14 large and distinct dorsolateral glandular warts; gular fold present; tips of fore and hind limbs overlapping when adpressed along the body; tips of fingers reaching between eye and nostril when foreleg is laid forward; dorsal surface and lateral sides of the head, upper and lower lips, dorsolateral glandular warts, vertebral ridge, the peripheral area of the cloaca and the ventral edge of the tail orange in coloration; the presence of a distinct black line extending from the posterior end of the eye towards the shoulder. Tylototritonngoclinhensissp. nov. is restricted to evergreen montane forests near water bodies on Ngoc Linh Mountain. We suggest that the new species should be classified as Endangered (EN) in the IUCN Red List. This new important discovery represents the eighth Tylototriton taxon described from Vietnam, and at the same time constitutes the southernmost distributional record for the whole genus in Asia.

Newtic1 Is a Component of Globular Structures That Accumulate along the Marginal Band of Erythrocytes in the Limb Blastema of Adult Newt, Cynops pyrrhogaster.

In adult newts, when a limb is amputated, a mesenchymal cell mass called the blastema is formed on the stump, where blood vessels filled with premature erythrocytes, named polychromatic normoblasts (PcNobs), elongate. We previously demonstrated that PcNobs in the blastema express an orphan gene, Newtic1, and that they secrete growth factors such as BMP2 and TGFß1 into the surrounding tissues. However, the relationship between Newtic1 expression and growth factor secretion was not clear since Newtic1 was thought to encode a membrane protein. In this study, we addressed this issue using morphological techniques and found that the Newtic1 protein is a component of globular structures that accumulate at the marginal band in the cytoplasm along the equator of PcNobs. Newtic1-positive (Newtic1(+)) globular structures along the equator were found only in PcNobs with a well-developed marginal band in the blastema. Newtic1(+) globular structures were associated with microtubules and potentially incorporated TGFß1. Based on these observations, we propose a hypothesis that the Newtic1 protein localizes to the membrane of secretory vesicles that primarily carry TGFß1 and binds to microtubules, thereby tethering secretory vesicles to microtubules and transporting them to the cell periphery as the marginal band develops.

Establishment of neural stem cell culture from the central nervous system of the Iberian ribbed newt Pleurodeles waltl.

Urodele amphibians have exceptional regeneration ability in various organs. Among these, the Iberian ribbed newt (Pleurodeles waltl) has emerged as a useful model organism for investigating the mechanisms underlying regeneration. Neural stem cells (NSCs) are an important source of regeneration in the central nervous system (CNS) and their culture method in vitro has been well established. NSCs form spherical cell aggregates called neurospheres and their formation has been demonstrated in various vertebrates, including some urodele species, but not in P. waltl. In this study, we reported neurosphere formation in brain- and spinal cord-derived cells of post-metamorphic P. waltl. These neurospheres showed proliferative activity and similar expression of marker proteins. However, the surface morphology was found to vary according to their origin, implying that the characteristics of the neurospheres generated from the brain and spinal cord could be similar but not identical. Subsequent in vitro differentiation analysis demonstrated that spinal cord-derived neurospheres gave rise to neurons and glial cells. We also found that cells in neurospheres from P. waltl differentiated to oligodendrocytes, whereas those from axolotls were reported not to differentiate to this cell type under standard culture conditions. Based on our findings, implantation of genetically modified neurospheres together with associated technical advantages in P. waltl could reveal pivotal gene(s) and/or signaling pathway(s) essential for the complete spinal cord regeneration ability in the future.

Identification of motilin in Japanese fire bellied newt.

Motilin, a peptide hormone consisting of 22 amino acid residues, was identified in the duodenum of pigs in the 1970s. It is known to induce gastrointestinal contractions during the interdigestive state in mammals. Although the motilin gene has been identified in various animal species, it has not been studied in amphibians. Here, we identified the motilin gene in the Japanese fire bellied newt (Cynops pyrrhogaster), and conducted an analysis of tissue distribution, morphological observations, and physiological experiments. The deduced mature newt motilin comprises 22 amino acid residues, like in mammals and birds. The C-terminus of the newt motilin showed high homology with motilin from other species compared to the N-terminus region, which is considered the bioactive site. Motilin mRNA expression in newts was abundant in the upper small intestine, with notably high motilin mRNA expression found in the pancreas. Motilin-producing cells were found in the mucosal layer of the upper small intestine and existed as two cell types: open-and closed-type cells. Motilin-producing cells in the pancreas were also found to produce insulin but not glucagon. Newt motilin stimulated gastric contractions but not in other parts of the intestines in vitro, and motilin-induced gastric contraction was significantly inhibited by treatment with atropine, a muscarinic acetylcholine receptor antagonist. These results indicate that motilin is also present in amphibians, and that its gastrointestinal contractile effects are conserved in mammals, birds, and amphibians. Additionally, we demonstrated for the first time the existence of pancreatic motilin, suggesting that newt motilin has an additional unknown physiological role.

Sustained Ranavirus Outbreak Causes Mass Mortality and Morbidity of Imperiled Amphibians in Florida.

A persistent 2-month long outbreak of Ranavirus in a natural community of amphibians contributed to a mass die-off of gopher frog tadpoles (Lithobates capito) and severe disease in striped newts (Notophthalmus perstriatus) in Florida. Ongoing mortality in L. capito and disease signs in N. perstriatus continued for 5 weeks after the first observation. Hemorrhagic disease and necrosis were diagnosed from pathological examination of L. capito tadpoles. We confirmed detection of a frog virus 3 (FV3)-like Ranavirus via quantitative PCR in all species. Our findings highlight the susceptibility of these species to Rv and the need for long-term disease surveillance during epizootics.

Development of the smooth newt, Lissotriton vulgaris, as observed in a garden pond during lockdown.

BACKGROUND: Lissotriton vulgaris ("smooth newt") was an important organism for experimental embryology, especially in the early 20th century. The stages of development of L. vulgaris from the appearance of the first cleavage to metamorphosis have previously been described, but only through drawings. RESULTS: Referring to Glaesner's staging and utilizing a makeshift lockdown laboratory, this study documents the development of L. vulgaris embryos in a small garden pond in Surrey, UK. This study describes varying developmental rates between individuals, identifies pigmented cells in close proximity/contributing to the heart, describes early gill development and reports a case of spontaneous, non-conjoined newt twins. CONCLUSIONS: While pigmentation in the heart has been reported in some amphibians, no pigmentation within urodele hearts has been documented to date. Furthermore, although the literature on conjoined twinning in amphibia is extensive, separate, non-conjoined twinning is largely unexplored in the literature. This study invites further research into understanding of these topics.